Heterologous production of 10-methylstearic acid by cells expressing recombinant methyltransferase

ABSTRACT

Disclosed herein are cells, nucleic acids, and proteins that can be used to produce branched (methyl)lipids, such as 10-methylstearic acids, and compositions that include such lipids. Cells disclosed herein comprise methyltransferase and/or reductase genes from bacteria of the class Gammaproteobacteria, which encode enzymes capable of catalyzing the production of branched (methyl)lipids from unbranched, unsaturated lipids. Saturated branched (methyl)lipids produced using embodiments of the present invention have favorable low-temperature fluidity and favorable oxidative stability, which are desirable properties for lubricants and specialty fluids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/648,117, filed Mar. 17, 2020, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2018/051919, filed Sep. 20, 2018, which claims the benefit ofpriority to U.S. Provisional Patent Application Ser. No. 62/561,136,filed Sep. 20, 2017, each of which are hereby incorporated by referencein their entirety.

This application is related to U.S. Ser. No. 15/710,734 andPCT/US17/52491 both filed Sep. 20, 2017.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referenceit its entirety. Said ASCII copy, created Jan. 30, 2022, is namedgibwp0006usc1_sequencelisting.txt and is 129 kilabytes in size.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention generally concerns production of branched (methyl)lipidsby cells expressing recombinant methyltransferases and/or reductasesderived from Gammaproteobacteria.

B. Description of Related Art

Fatty acids derived from agricultural plant and animal oils find use asindustrial lubricants, hydraulic fluids, greases, and other specialtyfluids in addition to oleochemical feedstocks for processing. Thephysical and chemical properties of these fatty acids result in largepart from their carbon chain length and number of unsaturated doublebonds. Fatty acids are typically 16:0 (sixteen carbons, zero doublebonds), 16:1 (sixteen carbons, 1 double bond), 18:0, 18:1, 18:2, or18:3. Importantly, fatty acids with no double bonds (saturated) havehigh oxidative stability, but they solidify at low temperature. Doublebonds improve low-temperature fluidity, but decrease oxidativestability. This trade-off poses challenges for lubricant and otherspecialty-fluid formulations because consistent long term performance(high oxidative stability) over a wide range of operating temperaturesis desirable. High 18:1 (oleic) fatty acid oils provide low temperaturefluidity with relatively good oxidative stability. Accordingly, severalcommercial products, such as high oleic soybean oil, high oleicsunflower oil, and high oleic algal oil, have been developed with higholeic compositions. Oleic acid is an alkene, however, and subject tooxidative degradation.

A superior alternative is the addition of a fully saturated methylbranch to the fatty acid chain. This creates a similarmelting-temperature depression as a double bond, but with no decrease inoxidative stability versus fully saturated linear fatty acids. Methylbranches located near the middle of the fatty acid chain have thelargest melting-temperature depression. Several chemical processes havebeen explored to introduce methyl branches; however, the preferredindustrial method results in random placement of the methyl branch andcreates a substantial amount of by-product. There remains a need forefficient and economical processes of producing branched (methyl)lipids.

SUMMARY OF THE INVENTION

Disclosed herein are cells, nucleic acids, and proteins that can be usedto produce branched (methyl) lipids, such as 10-methylstearic acids, andcompositions that include such lipids. Saturated branched (methyl)lipidsproduced using embodiments of the present invention have favorablelow-temperature fluidity and favorable oxidative stability, which aredesirable properties for lubricants and specialty fluids.

Various aspects relate to nucleic acids comprising a recombinant trnpBgene encoding a methyltransferase protein and/or a recombinant trnpAgene encoding a reductase protein. The methyltransferase protein and/orreductase protein may be proteins expressed by species of the classGammaproteobacteria (phylum, Proteobacteria), and the recombinant tmpBgene and/or recombinant tmpA gene may be codon-optimized for expressionin a different phylum of bacteria or in eukaryotes (e.g., yeast, such asArxula adeninivorans (also known as Blastobotrys adeninivorans orTrichosporon adeninivorans), Saccharomyces cerevisiae, or Yarrowialipolytica). The recombinant tmpB gene or recombinant tmpA gene may beoperably-linked to a promoter capable of driving expression in a phylumof bacteria other than Gammaproteobacteria or in eukaryotes (e.g.,yeast). The nucleic acid may be a plasmid or a chromosome.

Some aspects relate to a cell comprising a nucleic acid as describedherein. The cell may comprise a branched (methyl)lipid, such as10-methylstearic acid, and/or an exomethylene-substituted lipid, such as10-methylenestearic acid. The cell may be a eukaryotic cell, such as analgae cell, yeast cell, or plant cell.

Some aspects relate to a composition produced by cultivating a cellculture comprising cells as described herein. The oil composition maycomprise a branched (methyl)lipid, such as 10-methylstearic acid, and/oran exomethylene-substituted lipid, such as 10-methylenestearic acid. Insome embodiments, the oil composition is produced by cultivating a cellculture and recovering the oil composition from the cell culture,wherein the oil composition comprises 10-methyl fatty acids, and whereinthe 10-methyl fatty acids comprise at least about 1% by weight of thetotal fatty acids in the oil composition. In some embodiments, the20-methyl fatty acids comprise at least about 15% by weight of the totalfatty acids in the oil composition.

Some aspects relate to a method of producing an oil composition, themethod comprising: cultivating a cell culture comprising any of thecells disclosed herein; and recovering the oil composition from the cellculture. In some embodiments, the method further comprises contactingthe cell culture with a substrate comprising a fatty acid from 14 to 18carbons long with a double bond in the Δ9, Δ10, or Δ11 position. In someembodiments, recovering the oil composition from the cell culturecomprises recovering lipids that have been secreted by the cell. In someembodiments, producing the oil composition comprises performing chemicalreactions or causing chemical reactions to be performed in which oleicacid and methionine substrates are converted to 10-methylenestearicacid, wherein the chemical reactions are catalyzed by a tmpB protein. Insome embodiments, producing the oil composition comprises performingchemical reactions or causing chemical reactions to be performed inwhich 10-methylene stearic acid is reduced to 10-methylstearic acid,wherein the chemical reactions are catalyzed by tmpA protein. In someembodiments, the reduction is performed using NADPH, ferredoxin,flavodoxin, rubredoxin, cytochrome c, or combinations thereof asreducing agents. In any of the methods disclosed herein that involvereduction reactions any one of, or any combination of, NADPH,ferredoxin, flavodoxin, rubredoxin, and cytochrome c may be used.

Other objects, features and advantages of the present invention willbecome apparent from the following figures, detailed description, andexamples. It should be understood, however, that the figures, detaileddescription, and examples, while indicating specific embodiments of theinvention, are given by way of illustration only and are not meant to belimiting. Additionally, it is contemplated that changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one possible mechanism for the conversion of oleic acidto 10-methylstearic acid. An oleic acid substrate may be present as anacyl chain of a glycerolipid or phospholipid. A methionine substrate,which donates the methyl group, may be present as S-adenosyl methionine.The oleic acid and methionine substrates may be converted to10-methylenestearic acid (e.g., present as an acyl chain of aglycerolipid or phospholipid) and homocysteine (e.g., present asS-adenosyl homocysteine). This reaction may be catalyzed by a tmpBprotein as described herein, infra. 10-methylenestearic acid (e.g.,present as an acyl chain of a glycerolipid or phospholipid) may bereduced to 10-methylstearic acid. The reduction may be catalyzed by atmpA protein as describe herein, infra, for example, without limitation,using NADPH as a reducing agent. Other examples of the reducing agentmay include, without limitation, ferredoxin, flavodoxin, rubredoxin,cytochrome c, or combinations thereof. The language of the specificationand claims, however, is not limited to any particular reactionmechanism.

FIG. 2 shows the occurrence of cyclopropane fatty acyl phospholipidsynthase (cfa) homologs and 10-methylpalmitic acid (10Me16) in certainGammaproteobacteria with sequenced genomes and observed lipid profiles.

FIGS. 3A-3B depict maps of the following vectors, which encode a trnpoperon: pNC1071 (SEQ ID NO:39), which includes a Desulfobacter postgateitmp operon; pNC1072 (SEQ ID NO:40), which includes a Desulfobaculabalticum tmp operon, pNC1073 (SEQ ID NO:41), which includes aDesulfobacula toluolica tmp operon; pNC1074 (SEQ ID NO:42), whichincludes a Marinobacter hydrocarbonclasticus tmp operon; and pNC1076(SEQ ID NO:43), which includes a Thiohalospira halophila tmp operon.

FIG. 4 is a graph showing the percentage of 10-methylene fatty acids inSaccharomyces cerevisiae transformed with plasmids expressing tmpB fromthe indicated species: D. postgatei (D.po.), D. balticum (D.ba.), D.toluolica (D.to.), M. hydrocarbonclasticus (M.hy.) and T. halophila(T.ha.), or an empty vector control (-).

FIG. 5 is a graph showing the percentage of 10-methylene fatty acids inYarrowia lipolytica transformed with plasmids expressing tmpB from theindicated species: D. postgatei (D.po.), D. balticum (D.ba.), D.toluolica (D.to.), M. hydrocarbonclasticus (M.hy.) and T. halophila(T.ha.), or an empty vector control (-).

FIG. 6 shows the fatty acid profile of E. coli Top10 cells with plasmidspNC1071, pNC1072, pNC1073, pNC1074, pNC1076, and pNC53 (empty controlvector) grown in LB medium. Percentage values show the weight percent ofthe indicated fatty acid as a percentage of all fatty acids.14:0=Myristic acid, 16:0=Palmitic acid, 16:1Δ9=palmitoleic acid,16:0cyc=17Δ,cis-9,10-methylenehexadecanoic acid, 10-methylene16:0=10-methylene hexadecenoic acid, 18:1Δ11=vaccenic acid, 18:0=stearicacid, SD=standard deviation.

FIGS. 7A-7D show a CLUSTAL OMEGA alignment of tmpB protein sequencesencoded by the tmpB genes from Desulfobacula balticum, Marinobacterhydrocarbonclasticus, Thiohalospira halophila, Desulfobacter curvatus,Desulfobacter phenolica, Desulfobacula toluolica, Desulfobacterpostgatei, Halofilum ochraceum, and Marinobacter aquaeolei, along withthe cyclopropane fatty acid synthase (Cfa) enzyme from Escherichia coli.

FIGS. 8A-8D show a CLUSTAL OMEGA alignment tmpA protein sequencesencoded by the tmpA genes from Desulfobacula balticum, Marinobacterhydrocarbonclasticus, Thiohalospira halophila, Desulfobacter curvatus,Desulfobacter phenolica, Desulfobacula toluolica, Desulfobacterpostgatei, Halofilum ochraceum, and Marinobacter aquaeolei, along withthe Archaeoglobus fulgidus geranylgeranyl reductase protein AF0464.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “biologically-active portion” refers to an amino acid sequencethat is less than a full-length amino acid sequence, but exhibits atleast one activity of the full length sequence. For example, abiologically-active portion of a methyltransferase may refer to one ormore domains of tmpB having biological activity for converting oleicacid (e.g., a phospholipid comprising an ester of oleate) and methionine(e.g., S-adenosyl methionine) into 10-methylenestearic acid (e.g., aphospholipid comprising an ester of 10-methylenestearate). Abiologically-active portion of a reductase may refer to one or moredomains of tmpA having biological activity for converting10-methylenestearic acid (e.g., a phospholipid comprising an ester of10-methylenestearate) and a reducing agent (e.g., ferrodoxin,flavodoxin, rubredoxin, cytochrome c, NADH, NADPH, FAD, FADH₂, FMNH₂)into 10-methylstearic acid (e.g., a phospholipid comprising an ester of10-methylstearate). Biologically-active portions of a protein includepeptides or polypeptides comprising amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of the protein,e.g., the amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, that include feweramino acids than the full length protein, and exhibit at least oneactivity of the protein, especially methyltransferase or reductaseactivity. A biologically-active portion of a protein may comprise,comprise at least, or comprise at most, for example, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456,457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470,471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,499, 500, or more amino acids, or any range derivable therein.Typically, biologically-active portions comprise a domain or motifhaving a catalytic activity, such as catalytic activity for producing10-methylenestearic acid or 10-methylstearic acid. A biologically-activeportion of a protein includes portions of the protein that have the sameactivity as the full-length peptide and every portion that has moreactivity than background. For example, a biologically-active portion ofan enzyme may have, have at least, or have at most 0.1%, 0.5%, 1%, 2%,3%, 4%, 5%, 10%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100%, 100.1%, 100.2%,100.3%, 100.4%, 100.5%, 100.6%, 100.7%, 100.8%, 100.9%, 101%, 105%,110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%,190%, 200%, 220%, 240%, 260%, 280%, 300%, 320%, 340%, 360%, 380%, 400%or higher activity relative to the full-length enzyme (or any rangederivable therein). A biologically-active portion of a protein mayinclude portions of a protein that lack a domain that targets theprotein to a cellular compartment.

The terms “codon optimized” and “codon-optimized for the cell” refer tocoding nucleotide sequences (e.g., genes) that have been altered tosubstitute at least one codon that is relatively rare in a desired hostcell with a synonymous codon that is relatively prevalent in the hostcell. Codon optimization thereby allows for better utilization of thetRNA of a host cell by matching the codons of a recombinant gene withthe tRNA of the host cell. For example, the codon usage of the speciesof Gammaproteobacteria (prokaryotes) varies from the codon usage ofyeast (eukaryotes). The translation efficiency in a yeast host cell ofan mRNA encoding a Gammaproteobacteria protein may be increased bysubstituting the codons of the corresponding Gammaproteobacteria genewith codons that are more prevalent in the particular species of yeast.A codon optimized gene thereby has a nucleotide sequence that variesfrom a naturally-occurring gene.

The term “constitutive promoter” refers to a promoter that mediates thetranscription of an operably linked gene independent of a particularstimulus (e.g., independent of the presence of a reagent such asisopropyl β-D-1-thiogalactopyranoside).

The term “DGAT1” refers to a gene that encodes a type 1 diacylglycerolacyltransferase protein, such as a gene that encodes a yeast DGA2protein.

The term “DGAT2” refers to a gene that encodes a type 2 diacylglycerolacyltransferase protein, such as a gene that encodes a yeast DGA1protein.

“Diacylglyceride,” “diacylglycerol,” and “diglyceride,” are esterscomprised of glycerol and two fatty acids.

The terms “diacylglycerol acyltransferase” and “DGA” refer to anyprotein that catalyzes the formation of triacylglycerides fromdiacylglycerol. Diacylglycerol acyltransferases include type 1diacylglycerol acyltransferases (DGA2), type 2 diacylglycerolacyltransferases (DGA1), and type 3 diacylglycerol acyltransferases(DGA3) and all homologs that catalyze the above-mentioned reaction.

The terms “diacylglycerol acyltransferase, type 1” and “type 1diacylglycerol acyltransferases” refer to DGA2 and DGA2 orthologs.

The terms “diacylglycerol acyltransferase, type 2” and “type 2diacylglycerol acyltransferases” refer to DGA1 and DGA1 orthologs.

The term “domain” refers to a part of the amino acid sequence of aprotein that is able to fold into a stable three-dimensional structureindependent of the rest of the protein.

The term “drug” refers to any molecule that inhibits cell growth orproliferation, thereby providing a selective advantage to cells thatcontain a gene that confers resistance to the drug. Drugs includeantibiotics, antimicrobials, toxins, and pesticides.

“Dry weight” and “dry cell weight” mean weight determined in therelative absence of water. For example, reference to oleaginous cells ascomprising a specified percentage of a particular component by dry cellweight means that the percentage is calculated based on the weight ofthe cell after substantially all water has been removed. The term “% dryweight,” when referring to a specific fatty acid (e.g., oleic acid or10-methylstearic acid), includes fatty acids that are present ascarboxylates, esters, thioesters, and amides. For example, a cell thatcomprises 10-methylstearic acid as a percentage of total fatty acids by% dry cell weight includes 10-methylstearic acid, 10-methylstearate, the10-methylstearate portion of a diacylglycerol comprising a10-methylstearate ester, the 10-methylstearate portion of atriacylglycerol comprising a 10-methylstearate ester, the10-methylstearate portion of a phospholipid comprising a10-methylstearate ester, and the 10-methylstearate portion of10-methylstearate CoA. The term “% dry weight,” when referring to aspecific type of fatty acid (e.g., C16 fatty acids, C18 fatty acids),includes fatty acids that are present as carboxylates, esters,thioesters, and amides as described above (e.g., for 10 methylstearicacid).

The term “gene,” as used herein, may encompass genomic sequences thatcontain exons, particularly polynucleotide sequences encodingpolypeptide sequences involved in a specific activity. The term furtherencompasses synthetic nucleic acids that did not derive from genomicsequence. In certain embodiments, the genes lack introns, as they aresynthesized based on the known DNA sequence of cDNA and proteinsequence. In other embodiments, the genes are synthesized, non-nativecDNA wherein the codons have been optimized for expression in Y.lipolytica or A. adeninivorans based on codon usage. The term canfurther include nucleic acid molecules comprising upstream, downstream,and/or intron nucleotide sequences.

The term “inducible promoter” refers to a promoter that mediates thetranscription of an operably linked gene in response to a particularstimulus.

The term “integrated” refers to a nucleic acid that is maintained in acell as an insertion into the cell's genome, such as insertion into achromosome, including insertions into a plastid genome.

“In operable linkage” and “operably linked” refer to a functionallinkage between two nucleic acid sequences, such as a control sequence(typically a promoter) and the linked sequence (typically a sequencethat encodes a protein, also called a coding sequence). A promoter is inoperable linkage with a gene or is operably linked to a gene if it canmediate transcription of the gene.

The term “nucleic acid” refers to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. In allnucleic acid sequences provided herein, U nucleotides areinterchangeable with T nucleotides.

The term “phospholipid” refers to esters comprising glycerol, two fattyacids, and a phosphate. The phosphate may be covalently linked tocarbon-3 of the glycerol and comprise no further substitution, i.e., thephospholipid may be a phosphatidic acid. The phosphate may besubstituted with ethanolamine (e.g., phosphatidylethanolamine), choline(e.g., phosphatidylcholine), serine (e.g., phosphatidylserine), inositol(e.g., phosphatidylinositol), inositol phosphate (e.g.,phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate,phosphatidylinositol-5-phosphate), inositol bisphosphate (e.g.,phosphatidylinositol-4,5-bisphosphate), or inositol triphosphate (e.g.,phosphatidylinositol-3,4,5-bisphosphate).

As used herein, the term “plasmid” refers to a circular DNA moleculethat is physically separate from an organism's genomic DNA. Plasmids maybe linearized before being introduced into a host cell (referred toherein as a linearized plasmid). Linearized plasmids may not beself-replicating, but may integrate into and be replicated with thegenomic DNA of an organism.

A “promoter” is a nucleic acid control sequence that directs thetranscription of a nucleic acid. As used herein, a promoter includes thenecessary nucleic acid sequences near the start site of transcription.

The term “protein” refers to molecules that comprise an amino acidsequence, wherein the amino acids are linked by peptide bonds.

“Transformation” refers to the transfer of a nucleic acid into a hostorganism or into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid are referred to as “recombinant,” “transgenic,” or “transformed”organisms. Thus, nucleic acids of the present invention can beincorporated into recombinant constructs, typically DNA constructs,capable of introduction into and replication in a host cell. Such aconstruct can be a vector that includes a replication system andsequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell. Typically,expression vectors include, for example, one or more cloned genes underthe transcriptional control of 5′ and 3′ regulatory sequences and aselectable marker. Such vectors also can contain a promoter regulatoryregion (e.g., a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or location-specificexpression), a transcription initiation start site, a ribosome bindingsite, a transcription termination site, and/or a polyadenylation signal.

The term “transformed cell” refers to a cell that has undergone atransformation. Thus, a transformed cell comprises the parent's genomeand an inheritable genetic modification.

The terms “triacylglyceride,” “triacylglycerol,” “triglyceride,” and“TAG” are esters comprised of glycerol and three fatty acids.

The term “recombinant gene” refers to a gene that (1) is operativelylinked to a polynucleotide to which it is not linked in nature or (2)has a nucleotide sequence different from the naturally-occurringnucleotide sequence, such as, for example, a non-naturally occurringmutation, a codon-optimized sequence, or a cDNA that lacksnaturally-occurring introns that are found at the gene's genomic locus.The term “recombinant” can be used in reference to cloned DNA isolates,chemically synthesized polynucleotide analogs, or polynucleotide analogsthat are biologically synthesized by heterologous systems, as well asproteins and/or mRNAs encoded by such nucleic acids. Thus, for example,a protein synthesized by a microorganism is recombinant, if it issynthesized from an mRNA that is synthesized from a recombinant genepresent in the cell. As other examples, a gene may be a recombinant geneif it is operably linked to a promoter different from the promoter towhich it is operably linked in nature or if it is connected to anothergene or portion thereof and, together with the other gene or portionthereof, encodes a protein that is not found in nature, such as a fusionprotein or an epitope-tagged protein.

B. Microbe Engineering

1. Overview

Genes and gene products may be introduced into microbial host cells.Suitable host cells for expression of the genes and nucleic acidmolecules are microbial hosts that can be found broadly within thefungal or bacterial families. Examples of suitable host strains includebut are not limited to fungal or yeast species, such as Arxula,Aspegillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus,Cunninghamella, Hansenula, Kluyveromyces, Leucosporidiella, Lipomyces,Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium,Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon,Yarrowia, or bacterial species, such as members of proteobacteria andactinomycetes, as well as the genera Acinetobacter, Arthrobacter,Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces,Escherichia, Salmonella, Pseudomonas, and Cornyebacterium. Yarrowialipolytica and Arxula adeninivorans are suited for use as a hostmicroorganism because they can accumulate a large percentage of theirweight as triacylglycerols.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are known to those skilled in the art. Any of these could beused to construct chimeric genes to produce any one of the gene productsof the instant sequences. These chimeric genes could then be introducedinto appropriate microorganisms via transformation techniques to providehigh-level expression of the enzymes.

For example, a gene encoding an enzyme can be cloned in a suitableplasmid, and an aforementioned starting parent strain as a host can betransformed with the resulting plasmid. This approach can increase thecopy number of each of the genes encoding the enzymes and, as a result,the activities of the enzymes can be increased. The plasmid is notparticularly limited so long as it renders a desired geneticmodification inheritable to the microorganism's progeny.

Vectors or cassettes useful for the transformation of suitable hostcells are well known. Typically the vector or cassette containssequences that direct the transcription and translation of the relevantgene, a selectable marker, and sequences that allow autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene harboring transcriptional initiation controls anda region 3′ of the DNA fragment which controls transcriptionaltermination. In certain embodiments both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Promoters, cDNAs, and 3′ UTRs, as well as other elements of the vectors,can be generated through cloning techniques using fragments isolatedfrom native sources (see, e.g., Green & Sambrook, Molecular Cloning: ALaboratory Manual, (4th ed., 2012); U.S. Pat. No. 4,683,202(incorporated by reference)). Alternatively, elements can be generatedsynthetically using known methods (see, e.g., Gene 164:49-53 (1995)).

2. Homologous Recombination

Homologous recombination is the ability of complementary DNA sequencesto align and exchange regions of homology. Transgenic DNA (“donor”)containing sequences homologous to the genomic sequences being targeted(“template”) is introduced into the organism and then undergoesrecombination into the genome at the site of the correspondinghomologous genomic sequences.

The ability to carry out homologous recombination in a host organism hasmany practical implications for what can be carried out at the moleculargenetic level and is useful in the generation of a microbe that canproduce a desired product. By its nature homologous recombination is aprecise gene targeting event and, hence, most transgenic lines generatedwith the same targeting sequence will be essentially identical in termsof phenotype, necessitating the screening of far fewer transformationevents. Homologous recombination also targets gene insertion events intothe host chromosome, potentially resulting in excellent geneticstability, even in the absence of genetic selection. Because differentchromosomal loci will likely impact gene expression, even from exogenouspromoters/UTRs, homologous recombination can be a method of queryingloci in an unfamiliar genome environment and to assess the impact ofthese environments on gene expression.

A particularly useful genetic engineering approach using homologousrecombination is to co-opt specific host regulatory elements, such aspromoters/UTRs, to drive heterologous gene expression in a highlyspecific fashion.

Because homologous recombination is a precise gene targeting event, itcan be used to precisely modify any nucleotide(s) within a gene orregion of interest, so long as sufficient flanking regions have beenidentified. Therefore, homologous recombination can be used as a meansto modify regulatory sequences impacting gene expression of RNA and/orproteins. It can also be used to modify protein coding regions in aneffort to modify enzyme activities such as substrate specificity,affinities and Km, thereby affecting a desired change in the metabolismof the host cell. Homologous recombination provides a powerful means tomanipulate the host genome resulting in gene targeting, gene conversion,gene deletion, gene duplication, gene inversion, and exchanging geneexpression regulatory elements such as promoters, enhancers and 3′ UTRs.

Homologous recombination can be achieved by using targeting constructscontaining pieces of endogenous sequences to “target” the gene or regionof interest within the endogenous host cell genome. Such targetingsequences can either be located 5′ of the gene or region of interest, 3′of the gene/region of interest or even flank the gene/region ofinterest. Such targeting constructs can be transformed into the hostcell either as a supercoiled plasmid DNA with additional vectorbackbone, a PCR product with no vector backbone, or as a linearizedmolecule. In some cases, it may be advantageous to first expose thehomologous sequences within the transgenic DNA (donor DNA) by cuttingthe transgenic DNA with a restriction enzyme. This step can increase therecombination efficiency and decrease the occurrence of undesiredevents. Other methods of increasing recombination efficiency includeusing PCR to generate transforming transgenic DNA containing linear endshomologous to the genomic sequences being targeted.

3. Vectors and Vector Components

Vectors for transforming microorganisms in accordance with the presentinvention can be prepared by known techniques familiar to those skilledin the art in view of the disclosure herein. A vector typically containsone or more genes, in which each gene codes for the expression of adesired product (the gene product) and is operably linked to one or morecontrol sequences that regulate gene expression or target the geneproduct to a particular location in the recombinant cell.

a. Control Sequences

Control sequences are nucleic acids that regulate the expression of acoding sequence or direct a gene product to a particular location in oroutside a cell. Control sequences that regulate expression include, forexample, promoters that regulate transcription of a coding sequence andterminators that terminate transcription of a coding sequence. Anothercontrol sequence is a 3′ untranslated sequence located at the end of acoding sequence that encodes a polyadenylation signal. Control sequencesthat direct gene products to particular locations include those thatencode signal peptides, which direct the protein to which they areattached to a particular location inside or outside the cell.

Thus, an exemplary vector design for expression of a gene in a microbecontains a coding sequence for a desired gene product (for example, aselectable marker, or an enzyme) in operable linkage with a promoteractive in yeast. Alternatively, if the vector does not contain apromoter in operable linkage with the coding sequence of interest, thecoding sequence can be transformed into the cells such that it becomesoperably linked to an endogenous promoter at the point of vectorintegration. The promoter used to express a gene can be the promoternaturally linked to that gene or a different promoter.

A promoter can generally be characterized as constitutive or inducible.Constitutive promoters are generally active or function to driveexpression at all times (or at certain times in the cell life cycle) atthe same level. Inducible promoters, conversely, are active (or renderedinactive) or are significantly up- or down-regulated only in response toa stimulus. Both types of promoters find application in the methods ofthe invention. Inducible promoters useful in the invention include thosethat mediate transcription of an operably linked gene in response to astimulus, such as an exogenously provided small molecule, temperature(heat or cold), lack of nitrogen in culture media, etc. Suitablepromoters can activate transcription of an essentially silent gene orupregulate, e.g., substantially, transcription of an operably linkedgene that is transcribed at a low level.

Inclusion of termination region control sequence is optional, and ifemployed, then the choice is primarily one of convenience, as thetermination region is relatively interchangeable. The termination regionmay be native to the transcriptional initiation region (the promoter),may be native to the DNA sequence of interest, or may be obtainable fromanother source (See, e.g., Chen & Orozco, Nucleic Acids Research 16:8411(1988)).

b. Genes and Codon Optimization

Typically, a gene includes a promoter, a coding sequence, andtermination control sequences. When assembled by recombinant DNAtechnology, a gene may be termed an expression cassette and may beflanked by restriction sites for convenient insertion into a vector thatis used to introduce the recombinant gene into a host cell. Theexpression cassette can be flanked by DNA sequences from the genome orother nucleic acid target to facilitate stable integration of theexpression cassette into the genome by homologous recombination.Alternatively, the vector and its expression cassette may remainunintegrated (e.g., an episome), in which case, the vector typicallyincludes an origin of replication, which is capable of providing forreplication of the vector DNA.

A common gene present on a vector is a gene that codes for a protein,the expression of which allows the recombinant cell containing theprotein to be differentiated from cells that do not express the protein.Such a gene, and its corresponding gene product, is called a selectablemarker or selection marker. Any of a wide variety of selectable markerscan be employed in a transgene construct useful for transforming theorganisms of the invention.

For optimal expression of a recombinant protein, it is beneficial toemploy coding sequences that produce mRNA with codons optimally used bythe host cell to be transformed. Thus, proper expression of transgenescan require that the codon usage of the transgene matches the specificcodon bias of the organism in which the transgene is being expressed.The precise mechanisms underlying this effect are many, but include theproper balancing of available aminoacylated tRNA pools with proteinsbeing synthesized in the cell, coupled with more efficient translationof the transgenic messenger RNA (mRNA) when this need is met. When codonusage in the transgene is not optimized, available tRNA pools are notsufficient to allow for efficient translation of the transgenic mRNAresulting in ribosomal stalling and termination and possible instabilityof the transgenic mRNA. Resources for codon-optimization of genesequences are described in Puigbo et al., Nucleic Acids Research35:W126-31 (2007), and principles underlying codon optimizationstrategies are described in Angov, Biotechnology Journal 6:650-69(2011). Public databases providing statistics for codon usage bydifferent organisms are available, including at www.kazusa.or.jp/codon/and other publicly available databases and resources.

4. Transformation

Cells can be transformed by any suitable technique including, e.g.,biolistics, electroporation, glass bead transformation, and siliconcarbide whisker transformation. Any convenient technique for introducinga transgene into a microorganism can be employed in the presentinvention. Transformation can be achieved by, for example, the method ofD. M. Morrison (Methods in Enzymology 68:326 (1979)), the method byincreasing permeability of recipient cells for DNA with calcium chloride(Mandel & Higa, J. Molecular Biology, 53:159 (1970)), or the like.

Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowialipolytica) can be found in the literature (Bordes et al., J.Microbiological Methods, 70:493 (2007); Chen et al., AppliedMicrobiology & Biotechnology 48:232 (1997)). Examples of expression ofexogenous genes in bacteria such as E. coli are well known (Green &Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)).

Vectors for transformation of microorganisms in accordance with thepresent invention can be prepared by known techniques familiar to thoseskilled in the art. In one embodiment, an exemplary vector design forexpression of a gene in a microorganism contains a gene encoding anenzyme in operable linkage with a promoter active in the microorganism.Alternatively, if the vector does not contain a promoter in operablelinkage with the gene of interest, the gene can be transformed into thecells such that it becomes operably linked to a native promoter at thepoint of vector integration. The vector can also contain a second genethat encodes a protein. Optionally, one or both gene(s) is/are followedby a 3′ untranslated sequence containing a polyadenylation signal.Expression cassettes encoding the two genes can be physically linked inthe vector or on separate vectors. Co-transformation of microbes canalso be used, in which distinct vector molecules are simultaneously usedto transform cells (Protist 155:381-93 (2004)). The transformed cellscan be optionally selected based upon the ability to grow in thepresence of the antibiotic or other selectable marker under conditionsin which cells lacking the resistance cassette would not grow.

C. Exemplary Cells, Nucleic Acids, Compositions, and Methods

1. Transformed Cells

In some aspects, embodiments of the invention include cells transformedwith one or more nucleic acids encoding a methyltransferase and/orreductase protein. In some embodiments, the transformed cell is aprokaryotic cell, such as a bacterial cell. In some embodiments, thecell is a eukaryotic cell, such as a mammalian cell, a yeast cell, afilamentous fungi cell, a protist cell, an algae cell, an avian cell, aplant cell, or an insect cell. In some embodiments, the cell is a yeast.Those with skill in the art will recognize that many forms offilamentous fungi produce yeast-like growth, and the definition of yeastherein encompasses such cells. The cell may cell may be selected fromthe group consisting of algae, bacteria, molds, fungi, plants, andyeasts. The cell may be a yeast, fungus, or yeast-like algae. The cellmay be selected from thraustochytrids (Aurantiochytrium) andachlorophylic unicellular algae (Prototheca).

The cell may be selected from the group consisting of Arxula,Aspegillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus,Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea,Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca,Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces,Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, andYarrowia. It is specifically contemplated that one or more of these celltypes may be excluded from embodiments of this invention.

The cell may be selected from the group of consisting of Arxulaadeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillusterreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea,Cryptococcus albidus, Cryptococcus curvatus, Cryptococcusramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae,Cunninghamella echinulata, Cunninghamella japonica, Geotrichumfermentans, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromycesmarxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyceslipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierellaisabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii,Pichia guilliermondii, Pichia pastoris, Pichia stipites, Protothecazopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidiumtoruloides, Rhodosporidium paludigenum, Rhodotorula glutinis,Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomycespombe, Tremella enchepala, Trichosporon cutaneum, Trichosporonfermentans, Wickerhamomyces ciferrii, and Yarrowia lipolytica. It isspecifically contemplated that one or more of these cell types may beexcluded from embodiments of this invention.

In certain embodiments, the transformed cell comprises about, at leastabout, or at most about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or morelipid as measured by % dry cell weight, or any range derivable therein.In some embodiments, the transformed cell comprises C18 fatty acids at aconcentration of about, at least about, or at most about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or95% as a percentage of total C16 and C18 fatty acids in the cell byweight, or any range derivable therein.

In some embodiments, the transformed cell comprises oleic acid at aconcentration of about, at least about, or at most about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or higher as apercentage of total C16 and C18 fatty acids in the cell by weight, orany range derivable therein. In some embodiments, the transformed cellcomprises 10-methylstearic acid at a concentration of about, at leastabout, or of at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or higher as a percentageof total fatty acids in the cell by weight, or any range derivabletherein.

A cell may be modified to increase its oleate content, which serves as asubstrate for 10-methylstearate synthesis. Genetic modifications thatincrease oleate content are known (see, e.g., PCT Patent ApplicationPublication No. WO16/094520, published Jun. 16, 2016, herebyincorporated by reference in its entirety). For example, a cell maycomprise a 412 desaturase knockdown or knockout, which favors theaccumulation of oleate and disfavors the production of linoleate. A cellmay comprise a recombinant 49 desaturase gene, which favors theproduction of oleate and disfavors the accumulation of stearate. Therecombinant 49 desaturase gene may be, for example, the 49 desaturasegene from Y. lipolytica, Arxula adeninivorans, or Puccinia graminis. Acell may comprise a recombinant elongase 1 gene, which favors theproduction of oleate and disfavors the accumulation of palmitate andpalmitoleate. The recombinant elongase 1 gene may be the elongase 1 genefrom Y. lipolytica. A cell may comprise a recombinant elongase 2 gene,which favors the production of oleate and disfavors the accumulation ofpalmitate and palmitoleate. The recombinant elongase 2 gene may be theelongase 2 gene from R. norvegicus.

A cell may be modified to increase its triacylglycerol content, therebyincreasing its 10-methylstearate content. Genetic modifications thatincrease triacylglycerol content are known (see, e.g., PCT PatentApplication Publication No. WO16/094520, published Jun. 16, 2016, herebyincorporated by reference in its entirety). A cell may comprise arecombinant diacylglycerol acyltransferase gene (e.g., DGAT1, DGAT2, orDGAT3), which favors the production of triacylglycerols and disfavorsthe accumulation of diacylglycerols. The recombinant diacylglycerolacyltransferase gene may be, for example, DGAT2 (encoding protein DGA1)from Y. lipolytica, DGAT1 (encoding protein DGA2) from C. purpurea, orDGAT2 (encoding protein DGA1) from R. toruloides. The cell may comprisea glycerol-3-phosphate acyltransferase gene (Sct1) knockdown orknockout, which may favor the accumulation of triacylglycerols,depending on the cell type. The cell may comprise a recombinantglycerol-3-phosphate acyltransferase gene (Sct1) such as the Sct1 genefrom A. adeninivorans, which may favor the accumulation oftriacylglycerols. The cell may comprise a triacylglycerol lipase gene(TGL) knockdown or knockout, which may favor the accumulation oftriacylglycerols in the cell.

Various aspects of the invention relate to a transformed cell. Thetransformed cell may comprise a recombinant methyltransferase gene(e.g., a tmpB gene), a recombinant reductase gene (e.g., a tmpA gene),an exomethylene-substituted lipid, and/or a branched (methyl)lipid. Abranched (methyl)lipid may be a carboxylic acid (e.g., 10-methylstearicacid, 10-methylpalmitic acid, 12-methyloleic acid, 13-methyloleic acid,10-methyl-octadec-12-enoic acid), carboxylate (e.g., 10-methylstearate,10-methylpalmitate, 12-methyloleate, 13-methyloleate,10-methyl-octadec-12-enoate), ester (e.g., diacylglycerol,triacylglycerol, phospholipid), thioester (e.g., 10-methylstearyl CoA,10-methylpalmityl CoA, 12-methyloleoyl CoA, 13-methyloleoyl CoA,10-methyl-octadec-12-enoyl CoA), or amide. An exomethylene-substitutedlipid may be a carboxylic acid (e.g., 10-methylenestearic acid,10-methylenepalmitic acid, 12-methyleneoleic acid, 13-methyleneoleicacid, 10-methylene-octadec-12-enoic acid), carboxylate (e.g.,10-methylenestearate, 10-methylenepalmitate, 12-methyleneoleate,13-methyleneoleate, 10-methylene-octadec-12-enoate), ester (e.g.,diacylglycerol, triacylglycerol, phospholipid), thioester (e.g.,10-methylenestearyl CoA, 10-methylenepalmityl CoA, 12-methyleneoleoylCoA, 13-methyleneoleoyl CoA, 10-methylene-octadec-12-enoyl CoA), oramide. It is specifically contemplated that one or more of the abovelipids may be excluded from embodiments of this invention. Themethyltransferase gene and reductase gene may have the capability oftogether producing a methylated branch from any fatty acid from 14 to 18carbons long with an unsaturated double bond in the Δ9, Δ10, or Δ11position. The fatty acid may be 14, 15, 16, 17, or 18 carbons, or anyrange derivable therein.

“Fatty acids” generally exist in a cell as a phospholipid ortriacylglycerol, although they may also exist as a monoacylglycerol ordiacylglycerol, for example, as a metabolic intermediate. Free fattyacids also exist in the cell in equilibrium between a relativelyabundant carboxylate anion and a relatively scarce, neutrally-chargedacid. A fatty acid may exist in a cell as a thioester, especially as athioester with coenzyme A (CoA), during biosynthesis or oxidation. Afatty acid may exist in a cell as an amide, for example, when covalentlybound to a protein to anchor the protein to a membrane.

A cell may comprise any one of the nucleic acids described herein, infra(see, e.g., Section B, below). A cell may comprise multiple copies ofany one of the nucleic acids described herein. This can be accomplishedby, for example, including a tmpB and/or tmpB gene on a high-copy-numberplasmid that is transformed into a cell.

A branched (methyl)lipid may comprise a saturated branched aliphaticchain (e.g., 10-methylstearic acid, 10-methylpalmitic acid) or anunsaturated branched aliphatic chain (e.g., 12-methyloleic acid,13-methyloleic acid, 10-methyl-octadec-12-enoic acid). The branched(methyl)lipid may comprise a saturated or unsaturated branched aliphaticchain comprising a branching methyl group.

An exomethylene-substituted lipid may comprise a branched aliphaticchain (e.g., 10-methylenestearic acid, 10-methylenepalmitic acid,12-methyleneoleic acid, 13-methyleneoleic acid,10-methylene-octadec-12-enoic acid). The aliphatic chain may be branchedbecause the aliphatic chain is substituted with an exomethylene group.

A branched (methyl)lipid may be 10-methylstearate, or an acid(10-methylstearic acid), ester (e.g., diacylglycerol, triacylglycerol,phospholipid), thioester (e.g., 10-methylstearyl CoA), or amide (e.g.,10-methylstearyl amide) thereof. For example, the branched (methyl)lipidmay be a diacylglycerol, triacylglycerol, or phospholipid, and thediacylglycerol, triacylglycerol, or phospholipid may comprise an esterof 10-methylstearate.

An exomethylene-substituted lipid may be 10-methylenestearate, or anacid (10-methylenestearic acid), ester (e.g., diacylglycerol,triacylglycerol, phospholipid), thioester (e.g., 10-methylenestearylCoA), or amide (e.g., 10-methylenestearyl amide) thereof. For example,the exomethylene-substituted lipid may be a diacylglycerol,triacylglycerol, or phospholipid, and the diacylglycerol,triacylglycerol, or phospholipid may comprise an ester of10-methylenestearate.

In some embodiments, about, at least about, or at most about 1% of thefatty acids of the cell may be 10-methylstearic acid by weight. About,at least about, or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% by weight of the fatty acids of the cell maybe 10-methylstearic acid, or any range derivable therein.

In some embodiments, about, at least about, or at most about 1% of thefatty acids of the cell may be 10-methylenestearic acid by weight.About, at leat about, or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% by weight of the fatty acids of the cellmay be 10-methylenestearic acid, or any range derivable therein.

In some embodiments, about, at least about, or at most about 1% byweight of the fatty acids of the cell may be one or more of the branched(methyl)lipids described herein. About, at least about, or at most about2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weightof the fatty acids of the cell may be one or more of the branched(methyl)lipids described herein, or any range derivable therein.

In some embodiments, about, at least about, or at most about 1% byweight of the fatty acids of the cell may be one or more of the branched(methyl)lipids described herein. About, at least about, or at most about2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weightof the fatty acids of the cell may one or more of the branched(methyl)lipids described herein, or any range derivable therein.

In some embodiments, the cell may comprise about, at least about, or atmost about 1% 10-methylstearic acid as measured by % dry cell weight.The cell may comprise about, at least about, or at most about 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, or 50% 10-methylstearic acid as measured by % dry cellweight, or any range derivable therein.

In some embodiments, the cell may comprise about, at least about, or atmost about 1% 10-methylenestearic acid as measured by % dry cell weight.The cell may comprise about, at least about, or at most about 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, or 50% 10-methylenestearic acid as measured by % dry cellweight, or any range derivable therein.

An unmodified cell of the same type (e.g., species) as a cell of theinvention may not comprise 10-methylstearate, or an acid(10-methylstearic acid), ester (e.g., diacylglycerol, triacylglycerol,phospholipid), thioester (e.g., 10-methylstearyl CoA), or amide (e.g.,10-methylstearyl amide) thereof (e.g., wherein the unmodified cell doesnot comprise a recombinant methyltransferase gene or a recombinantreductase gene). An unmodified cell of the same type (e.g., species) asa cell of the invention may not comprise 10-methylenestearate, or anacid (10-methylenestearic acid), ester (e.g., diacylglycerol,triacylglycerol, phospholipid), thioester (e.g., 10-methylenestearylCoA), or amide (e.g., 10-methylenestearyl amide) thereof (e.g., whereinthe unmodified cell does not comprise a recombinant methyltransferasegene or a recombinant reductase gene). In some embodiments, anunmodified cell of the same species as the cell does not comprise abranched (methyl)lipid and/or an exomethylene-substituted lipid. In someembodiments, an unmodified cell of the same species as the cell does notcomprise one or more of the branched (methyl)lipids orexomethylene-substituted lipids described herein.

In some embodiments, a cell may constitutively express the proteinencoded by a recombinant methyltransferase gene and/or reductase gene. Acell may constitutively express a methyltransferase protein and/orreductase protein.

2. Nucleic Acids

a. General

Various aspects of the invention relate to a nucleic acid comprising arecombinant methyltransferase gene, a recombinant reductase gene, orboth. The nucleic acid may be, for example, a plasmid. In someembodiments, a recombinant methyltransferase gene and/or a recombinantreductase gene is integrated into the genome of a cell, and thus, thenucleic acid may be a chromosome. In some embodiments, the inventionrelates to a cell comprising a recombinant methyltransferase gene, e.g.,wherein the recombinant methyltransferase gene is present in a plasmidor chromosome. In some embodiments, the invention relates to a cellcomprising a recombinant reductase gene, e.g., wherein the recombinantreductase gene is present in a plasmid or chromosome. A recombinantmethyltransferase gene and a recombinant reductase gene may be presentin a cell in the same nucleic acid (e.g., same plasmid or chromosome) orin different nucleic acids (e.g., different plasmids or chromosomes).

A nucleic acid may be inheritable to the progeny of a transformed cell.A gene such as a recombinant methyltransferase gene or recombinantreductase gene may be inheritable because it resides on a plasmid orchromosome. In certain embodiments, a gene may be inheritable because itis integrated into the genome of the transformed cell.

A gene may comprise conservative substitutions, deletions, and/orinsertions while still encoding a protein that has activity. Forexample, codons may be optimized for a particular host cell, differentcodons may be substituted for convenience, such as to introduce arestriction site or to create optimal PCR primers, or codons may besubstituted for another purpose. Similarly, the nucleotide sequence maybe altered to create conservative amino acid substitutions, deletions,and/or insertions.

Proteins may comprise conservative substitutions, deletions, and/orinsertions while still maintaining activity. Conservative substitutiontables are well known in the art (Creighton, Proteins (2d. ed., 1992)).

Amino acid substitutions, deletions and/or insertions may readily bemade using recombinant DNA manipulation techniques. Methods for themanipulation of DNA sequences to produce substitution, insertion ordeletion variants of a protein are well known in the art. These methodsinclude M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland,Ohio), Quick Change Site Directed mutagenesis (Stratagene, San Diego,Calif.), PCR-mediated site-directed mutagenesis, and other site-directedmutagenesis protocols.

A “coding sequence” or “coding region” refers to a nucleic acid moleculehaving sequence information necessary to produce a protein product, suchas an amino acid or polypeptide, when the sequence is expressed. Thecoding sequence may comprise and/or consist of untranslated sequences(including introns or 5′ or 3′ untranslated regions) within translatedregions, or may lack such intervening untranslated sequences (e.g., asin cDNA).

The abbreviation used throughout the specification to refer to nucleicacids comprising and/or consisting of nucleotide sequences are theconventional one-letter abbreviations. Thus, when included in a nucleicacid, the naturally occurring encoding nucleotides are abbreviated asfollows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil(U). Also, unless otherwise specified, the nucleic acid sequencespresented herein is the 5′→3′ direction.

b. Nucleic Acids Comprising a Recombinant Methyltransferase Gene

A methyltransferase gene (e.g., a recombinant methyltransferase gene)encodes a methyltransferase protein, which is an enzyme capable oftransferring a carbon atom and one or more protons bound thereto from asubstrate such as S-adenosyl methionine to a fatty acid such as oleicacid (e.g., wherein the fatty acid is present as a free fatty acid,carboxylate, phospholipid, diacylglycerol, or triacylglycerol). Themethyltransferase gene (e.g., a recombinant methyltransferase gene) mayhave a coding region that is identical to one from a bacterium of theclass Gammaproteobacteria. The methyltransferase gene may comprise anyone of the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, and SEQ ID NO:17. The methyltransferase gene (e.g., arecombinant methyltransferase gene) may be a 10-methylstearic B gene(tmpB) as described herein, or a biologically-active portion thereof(i.e., wherein the biologically-active portion thereof comprisesmethyltransferase activity).

The methyltransferase gene (e.g., a recombinant methyltransferase gene)may be derived from a species of Gammaproteobacteria, such as bacteriafrom the genera Desulfobacter, Desulfobacula, Marinobacter,Thiohalospira, or Halofilum. The methyltransferase gene (e.g., arecombinant methyltransferase gene) may be selected from the groupconsisting of Desulfobacula balticum gene tmpB (SEQ ID NO:1),Marinobacter hydrocarbonclasticus gene tmpB (SEQ ID NO:3), Thiohalospirahalophila gene tmpB (SEQ ID NO:5), Desulfobacter curvatus gene tmpB (SEQID NO:7), Desulfobacter phenolica gene tmpB (SEQ ID NO:9), Desulfobaculatoluolica gene tmpB (SEQ ID NO:11), Desulfobacter postgatei gene tmpB(SEQ ID NO:13), Halofilum ochraceum gene tmpB (SEQ ID NO:15), andMarinobacter aquaeolei gene tmpB (SEQ ID NO:17). It is specificallycontemplated that one or more of the above methyltransferase genes maybe excluded from embodiments of this invention.

A recombinant methyltransferase gene may be recombinant because it isoperably linked to a promoter other than the naturally-occurringpromoter of the methyltransferase gene. Such genes may be useful todrive transcription in a particular species of cell. A recombinantmethyltransferase gene may be recombinant because it contains one ormore nucleotide substitutions relative to a naturally-occurringmethyltransferase gene. Such genes may be useful to increase thetranslation efficiency of the methyltransferase gene's mRNA transcriptin a particular species of cell.

A nucleic acid may comprise a recombinant methyltransferase gene and apromoter, wherein the recombinant methyltransferase gene and promoterare operably linked. The recombinant methyltransferase gene and promotermay be derived from different species. For example, the recombinantmethyltransferase gene may encode the methyltransferase protein of aspecies of Gammaproteobacteria, and the recombinant methyltransferasegene may be operably-linked to a promoter that can drive transcriptionin another type of bacteria or a eukaryote (e.g., an algae cell, yeastcell, or plant cell). The promoter may be a eukaryotic promoter. A cellmay comprise the nucleic acid, and the promoter may be capable ofdriving transcription in the cell. A cell may comprise a recombinantmethyltransferase gene, and the recombinant methyltransferase gene maybe operably linked to a promoter capable of driving transcription of therecombinant methyltransferase gene in the cell. The cell may be aspecies of yeast, and the promoter may be a yeast promoter. The cell maybe a species of bacteria, and the promoter may be a bacterial promoter(e.g., wherein the bacterial promoter is not a promoter from aGammaproteobacterium). The cell may be a species of algae, and thepromoter may be an algae promoter. The cell may be a species of plant,and the promoter may be a plant promoter.

A recombinant methyltransferase gene may be operably linked to apromoter that cannot drive transcription in the cell from which therecombinant methyltransferase gene originated. For example, the promotermay not be capable of binding an RNA polymerase of the cell from which arecombinant methyltransferase gene originated. In some embodiments, thepromoter cannot bind a prokaryotic RNA polymerase and/or initiatetranscription mediated by a prokaryotic RNA polymerase. In someembodiments, a recombinant methyltransferase gene is operably-linked toa promoter that cannot drive transcription in the cell from which theprotein encoded by the gene originated. For example, the promoter maynot be capable of binding an RNA polymerase of a cell that naturallyexpresses the methyltransferase enzyme encoded by a recombinantmethyltransferase gene.

A promoter may be an inducible promoter or a constitutive promoter. Apromoter may be any one of the promoters described in PCT PatentApplication Publication No. WO 2016/014900, published Jan. 28, 2016(hereby incorporated by reference in its entirety). WO 2016/014900describes various promoters derived from yeast species Yarrowialipolytica and Arxula adeninivorans, which may be particularly useful aspromoters for driving the transcription of a recombinant gene in a yeastcell. A promoter may be a promoter from a gene encoding a TranslationElongation factor EF-1α; Glycerol-3-phosphate dehydrogenase;Triosephosphate isomerase 1; Fructose-1,6-bisphosphate aldolase;Phosphoglycerate mutase; Pyruvate kinase; Export protein EXP1; Ribosomalprotein S7; Alcohol dehydrogenase; Phosphoglycerate kinase; HexoseTransporter; General amino acid permease; Serine protease; Isocitratelyase; Acyl-CoA oxidase; ATP-sulfurylase; Hexokinase; 3-phosphoglyceratedehydrogenase; Pyruvate Dehydrogenase Alpha subunit; PyruvateDehydrogenase Beta subunit; Aconitase; Enolase; Actin; Multidrugresistance protein (ABC-transporter); Ubiquitin; GTPase; Plasma membraneNa+/P_(i) cotransporter; Pyruvate decarboxylase; Phytase; orAlpha-amylase, e.g., wherein the gene is a yeast gene, such as a genefrom Yarrowia lipolytica or Arxula adeninivorans.

A recombinant methyltransferase gene may comprise a nucleotide sequencewith, with at least, or with at most 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity with the nucleotide sequence set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17. A recombinantmethyltransferase gene may comprise a nucleotide sequence with, with atleast, or with at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity with 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or1300 contiguous base pairs of the nucleotide sequence set forth in SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17. A recombinantmethyltransferase may or may not have 100% sequence identity with anyone of the nucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, or SEQ ID NO:17. A recombinant methyltransferase gene may ormay not have 100% sequence identity with 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, or 1300 contiguous base pairs starting at nucleotideposition 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456,457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470,471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512,513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526,527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540,541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568,569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582,583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596,597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610,611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638,639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652,653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694,695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708,709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722,723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736,737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750,751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764,765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778,779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792,793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806,807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820,821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834,835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848,849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862,863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876,877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890,891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904,905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918,919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932,933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946,947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960,961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974,975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988,989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002,1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014,1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026,1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038,1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050,1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062,1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074,1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086,1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098,1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110,1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122,1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134,1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146,1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158,1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170,1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182,1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194,1195, 1196, 1197, 1198, 1199, or 1200 of the nucleotide sequence setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17. Arecombinant methyltransferase gene may comprise a nucleotide sequencewith, with at least, or with at most 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity with the nucleotide sequence set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17, and the recombinantmethyltransferase gene may encode a methyltransferase protein with, withat least, or with at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with the amino acid sequence set forth in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, or SEQ ID NO:18. For example, a gene that iscodon-optimized for expression in yeast may have about 70% sequenceidentity with SEQ ID NO:1, while the protein encoded by such acodon-optimized gene may have 100% sequence identity with the amino acidsequence set forth in SEQ ID NO:2. Thus, even though a codon-optimizedgene may have only about 70% sequence identity or less to the originalgene, the codon-optimized gene encodes the same amino acid sequence ofthe original gene.

A recombinant methyltransferase gene may vary from a naturally-occurringmethyltransferase gene because the recombinant methyltransferase genemay be codon-optimized for expression in a eukaryotic cell, such as aplant cell, algae cell, or yeast cell. A cell may comprise a recombinantmethyltransferase gene, wherein the recombinant methyltransferase geneis codon-optimized for the cell.

Exactly, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,495, 496, 497, 498, 499, or 500 codons of a recombinantmethyltransferase gene may vary from a naturally-occurringmethyltransferase gene or may be unchanged from a naturally-occurringmethyltransferase gene. For example, a recombinant methyltransferasegene may comprise a nucleotide sequence with at least about 65% sequenceidentity with the naturally-occurring nucleotide sequence set forth inSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17 (e.g., at least about70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity),and at least 5 codons of the nucleotide sequence of the recombinantmethyltransferase gene may vary from the naturally-occurring nucleotidesequence (e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, or 100 codons).

A methyltransferase gene encodes a methyltransferase protein. Amethyltransferase protein may be a protein expressed by a species ofGammaproteobacteria, such as bacteria from the genera Desulfobacter,Desulfobacula, Marinobacter, Thiohalospira, or Halofilum. A recombinantmethyltransferase gene may encode a naturally-occurringmethyltransferase protein even if the recombinant methyltransferase geneis not a naturally-occurring methyltransferase gene. For example, arecombinant methyltransferase gene may vary from a naturally-occurringmethyltransferase gene because the recombinant methyltransferase gene iscodon-optimized for expression in a specific cell. The codon-optimized,recombinant methyltransferase gene and the naturally-occurringmethyltransferase gene may nevertheless encode the samenaturally-occurring methyltransferase protein.

A recombinant methyltransferase gene may encode a methyltransferaseprotein selected from the group consisting of Desulfobacula balticumprotein tmpB (SEQ ID NO:2), Marinobacter hydrocarbonclasticus proteintmpB (SEQ ID NO:4), Thiohalospira halophila protein tmpB (SEQ ID NO:6),Desulfobacter curvatus protein tmpB (SEQ ID NO:8), Desulfobacterphenolica protein tmpB (SEQ ID NO:10), Desulfobacula toluolica proteintmpB (SEQ ID NO:12), Desulfobacter postgatei protein tmpB (SEQ IDNO:14), Halofilum ochraceum protein tmpB (SEQ ID NO:16), andMarinobacter aquaeolei protein tmpB (SEQ ID NO:18). It is specificallycontemplated that one or more of the above methyltransferase proteinsmay be excluded from embodiments of this invention. A recombinantmethyltransferase gene may encode a methyltransferase protein, and themethyltransferase protein may be substantially identical to any one ofthe foregoing enzymes, but the recombinant methyltransferase gene mayvary from the naturally-occurring gene that encodes the enzyme. Therecombinant methyltransferase gene may vary from the naturally-occurringgene because the recombinant methyltransferase gene may becodon-optimized for expression in a specific phylum, class, order,family, genus, species, or strain of cell.

The sequences of naturally-occurring methyltransferase proteins are setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18. Arecombinant methyltransferase gene may or may not encode a proteincomprising 100% sequence identity with the amino acid sequence set forthin SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18. For example, arecombinant methyltransferase gene may encode a protein having 100%sequence identity with a biologically-active portion of an amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ IDNO:18.

A recombinant methyltransferase gene may encode a methyltransferaseprotein having, having at least, or having at most 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 100% sequence identity with the amino acid sequence setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18, or abiologically-active portion thereof. A recombinant methyltransferasegene may encode a methyltransferase protein having about, at leastabout, or at most about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, 100%, 100.1%, 100.2%, 100.3%, 100.4%, 100.5%,100.6%, 100.7%, 100.8%, 100.9%, 101%, 105%, 110%, 115%, 120%, 125%,130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 220%, 240%,260%, 280%, 300%, 320%, 340%, 360%, 380%, or 400% methyltransferaseactivity relative to a protein comprising the amino acid sequence setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18. Arecombinant methyltransferase gene may encode a protein having at least65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, or 500 contiguous amino acids starting at position1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500of the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, or SEQ ID NO:18.

Substrates for the methyltransferase protein may include any fatty acidfrom 14 to 18 carbons long with an unsaturated double bond in the Δ9,Δ10, or Δ11 position. The substrate may have a chain that is 14, 15, 16,17, or 18 carbons long, or any range derivable therein. Themethyltransferase protein may be capable of catalyzing the formation ofa methylene substitution at the Δ9, Δ10, or Δ11 position of such asubstrate.

In some embodiments, the recombinant methyltransferase gene encodes amethyltransferase protein that has specific amino acids unchanged fromthe amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, or SEQ ID NO:18. The unchanged amino acids can include 1, 2, 3,4, 5, 6, 7, 8, or 9 amino acids selected from Y163, T175, R199, E211,G269, Y271, N313, N319, and W389 of Marinobacter hydrocarbonclasticustmpB or corresponding amino acids in tmpB from Desulfobacula balticum,Thiohalospira halophila, Desulfobacter curvatus, Desulfobacterphenolica, Desulfobacula toluolica, Desulfobacter postgatei, Halofilumochraceum, or Marinobacter aquaeolei, according to the alignment setforth in FIGS. 7A-D.

c. Nucleic Acids Comprising a Recombinant Reductase Gene

A reductase gene (e.g., a recombinant reductase gene) encodes areductase protein, which is an enzyme capable of reducing a double bondof a fatty acid (e.g., wherein the fatty acid is present as a free fattyacid, carboxylate, phospholipid, diacylglycerol, or triacylglycerol).The reductase gene (e.g., a recombinant reductase gene) may have acoding region that is identical to one from a bacterium of the classGammaproteobacteria. The reductase gene may comprise any one of thenucleotide sequences set forth in SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, and SEQ ID NO:35. The reductase gene (e.g., a recombinantreductase gene) may be a 10-methylstearic A gene (tmpA) as describedherein, or a biologically-active portion thereof (i.e., wherein thebiologically-active portion thereof comprises reductase activity).

The reductase gene (e.g., a recombinant reductase gene) may be derivedfrom a species of Gammaproteobacteria, such as bacteria from the generaDesulfobacter, Desulfobacula, Marinobacter, Thiohalospira, or Halofilum.The reductase gene (e.g., a recombinant reductase gene) may be selectedfrom the group consisting of Desulfobacula balticum gene tmpA (SEQ IDNO:19), Marinobacter hydrocarbonclasticus gene tmpA (SEQ ID NO:21),Thiohalospira halophila gene tmpA (SEQ ID NO:23), Desulfobacter curvatusgene tmpA (SEQ ID NO:25), Desulfobacter phenolica gene tmpA (SEQ IDNO:27), Desulfobacula toluolica gene tmpA (SEQ ID NO:29), Desulfobacterpostgatei gene tmpA (SEQ ID NO:31), Halofilum ochraceum gene tmpA (SEQID NO:33), and Marinobacter aquaeolei gene tmpA (SEQ ID NO:35). It isspecifically contemplated that one or more of the above reductase genesmay be excluded from embodiments of this invention.

A recombinant reductase gene may be recombinant because it is operablylinked to a promoter other than the naturally-occurring promoter of thereductase gene. Such genes may be useful to drive transcription in aparticular species of cell. A recombinant reductase gene may berecombinant because it contains one or more nucleotide substitutionsrelative to a naturally-occurring reductase gene. Such genes may beuseful to increase the translation efficiency of the reductase gene'smRNA transcript in a particular species of cell.

A nucleic acid may comprise a recombinant reductase gene and a promoter,wherein the recombinant reductase gene and promoter are operably linked.The recombinant reductase gene and promoter may be derived fromdifferent species. For example, the recombinant reductase gene mayencode the reductase protein of a species of Gammaproteobacteria, andthe recombinant reductase gene may be operably-linked to a promoter thatcan drive transcription in another type of bacteria or a eukaryote(e.g., an algae cell, yeast cell, or plant cell). The promoter may be aeukaryotic promoter. A cell may comprise the nucleic acid, and thepromoter may be capable of driving transcription in the cell. A cell maycomprise a recombinant reductase gene, and the recombinant reductasegene may be operably linked to a promoter capable of drivingtranscription of the recombinant reductase gene in the cell. The cellmay be a species of yeast, and the promoter may be a yeast promoter. Thecell may be a species of bacteria, and the promoter may be a bacterialpromoter (e.g., wherein the bacterial promoter is not a promoter from aGammaproteobacterium). The cell may be a species of algae, and thepromoter may be an algae promoter. The cell may be a species of plant,and the promoter may be a plant promoter.

A recombinant reductase gene may be operably linked to a promoter thatcannot drive transcription in the cell from which the recombinantreductase gene originated. For example, the promoter may not be capableof binding an RNA polymerase of the cell from which a recombinantreductase gene originated. In some embodiments, the promoter cannot binda prokaryotic RNA polymerase and/or initiate transcription mediated by aprokaryotic RNA polymerase. In some embodiments, a recombinant reductasegene is operably-linked to a promoter that cannot drive transcription inthe cell from which the protein encoded by the gene originated. Forexample, the promoter may not be capable of binding an RNA polymerase ofa cell that naturally expresses the reductase enzyme encoded by arecombinant reductase gene.

A promoter may be an inducible promoter or a constitutive promoter. Apromoter may be any one of the promoters described in PCT PatentApplication Publication No. WO 2016/014900, published Jan. 28, 2016(hereby incorporated by reference in its entirety). WO 2016/014900describes various promoters derived from yeast species Yarrowialipolytica and Arxula adeninivorans, which may be particularly useful aspromoters for driving the transcription of a recombinant gene in a yeastcell. A promoter may be a promoter from a gene encoding a TranslationElongation factor EF-1α; Glycerol-3-phosphate dehydrogenase;Triosephosphate isomerase 1; Fructose-1,6-bisphosphate aldolase;Phosphoglycerate mutase; Pyruvate kinase; Export protein EXP1; Ribosomalprotein S7; Alcohol dehydrogenase; Phosphoglycerate kinase; HexoseTransporter; General amino acid permease; Serine protease; Isocitratelyase; Acyl-CoA oxidase; ATP-sulfurylase; Hexokinase; 3-phosphoglyceratedehydrogenase; Pyruvate Dehydrogenase Alpha subunit; PyruvateDehydrogenase Beta subunit; Aconitase; Enolase; Actin; Multidrugresistance protein (ABC-transporter); Ubiquitin; GTPase; Plasma membraneNa+/P_(i) cotransporter; Pyruvate decarboxylase; Phytase; orAlpha-amylase, e.g., wherein the gene is a yeast gene, such as a genefrom Yarrowia lipolytica or Arxula adeninivorans.

A recombinant reductase gene may comprise a nucleotide sequence with,with at least, or with at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with the nucleotide sequence set forth in SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35. A recombinantreductase gene may comprise a nucleotide sequence with, with at least,or with at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity with 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or1300 contiguous base pairs starting at nucleotide position 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278,279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488,489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502,503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530,531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544,545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558,559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586,587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600,601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614,615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628,629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642,643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656,657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670,671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684,685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698,699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712,713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726,727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740,741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754,755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768,769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782,783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796,797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810,811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824,825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838,839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852,853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880,881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894,895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908,909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922,923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936,937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950,951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964,965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978,979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992,993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005,1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017,1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029,1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041,1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053,1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065,1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077,1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089,1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101,1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113,1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125,1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137,1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149,1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161,1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173,1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185,1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197,1198, 1199, 1200 of the nucleotide sequence set forth in SEQ ID NO:19,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35. A recombinant reductase mayor may not have 100% sequence identity with any one of the nucleotidesequences set forth in SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, and SEQID NO:35. A recombinant reductase gene may or may not have 100% sequenceidentity with 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or1300 contiguous base pairs of the nucleotide sequence set forth in SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35. A recombinantreductase gene may comprise a nucleotide sequence with, with at least,or with at most 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity with the nucleotide sequence set forth in SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, or SEQ ID NO:35, and the recombinant reductase genemay encode a reductase protein with, with at least, or with at most 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acidsequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ IDNO:36. For example, a gene that is codon-optimized for expression inyeast may have about 70% sequence identity with SEQ ID NO:19, while theprotein encoded by such a codon-optimized gene may have 100% sequenceidentity with the amino acid sequence set forth in SEQ ID NO:20. Thus,even though a codon-optimized gene may have only about 70% sequenceidentity or less to the original gene, the codon-optimized gene encodesthe same amino acid sequence of the original gene.

A recombinant reductase gene may vary from a naturally-occurringreductase gene because the recombinant reductase gene may becodon-optimized for expression in a eukaryotic cell, such as a plantcell, algae cell, or yeast cell. A cell may comprise a recombinantreductase gene, wherein the recombinant reductase gene iscodon-optimized for the cell.

Exactly, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,495, 496, 497, 498, 499, or 500 codons of a recombinant reductase genemay vary from a naturally-occurring reductase gene or may be unchangedfrom a naturally-occurring reductase gene. For example, a recombinantreductase gene may comprise a nucleotide sequence with at least about65% sequence identity with the naturally-occurring nucleotide sequenceset forth in SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35(e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% sequence identity), and at least 5 codons of the nucleotide sequenceof the recombinant reductase gene may vary from the naturally-occurringnucleotide sequence (e.g., at least about 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70, 80, 90, or 100 codons).

A reductase gene encodes a reductase protein. A reductase protein may bea protein expressed by a species of Gammaproteobacteria, such asbacteria from the genera Desulfobacter, Desulfobacula, Marinobacter,Thiohalospira, or Halofilum. A recombinant reductase gene may encode anaturally-occurring reductase protein even if the recombinant reductasegene is not a naturally-occurring reductase gene. For example, arecombinant reductase gene may vary from a naturally-occurring reductasegene because the recombinant reductase gene is codon-optimized forexpression in a specific cell. The codon-optimized, recombinantreductase gene and the naturally-occurring reductase gene maynevertheless encode the same naturally-occurring reductase protein.

A recombinant reductase gene may encode a reductase protein selectedfrom the group consisting of Desulfobacula balticum protein tmpA (SEQ IDNO:20), Marinobacter hydrocarbonclasticus protein tmpA (SEQ ID NO:22),Thiohalospira halophila protein tmpA (SEQ ID NO:24), Desulfobactercurvatus protein tmpA (SEQ ID NO:26), Desulfobacter phenolica proteintmpA (SEQ ID NO:28), Desulfobacula toluolica protein tmpA (SEQ IDNO:30), Desulfobacter postgatei protein tmpA (SEQ ID NO:32), Halofilumochraceum protein tmpA (SEQ ID NO:34), and Marinobacter aquaeoleiprotein tmpA (SEQ ID NO:36). It is specifically contemplated that one ormore of the above reductase proteins may be excluded from embodiments ofthis invention. A recombinant reductase gene may encode a reductaseprotein, and the reductase protein may be substantially identical to anyone of the foregoing enzymes, but the recombinant reductase gene mayvary from the naturally-occurring gene that encodes the enzyme. Therecombinant reductase gene may vary from the naturally-occurring genebecause the recombinant reductase gene may be codon-optimized forexpression in a specific phylum, class, order, family, genus, species,or strain of cell.

The sequences of naturally-occurring reductase proteins are set forth inSEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36. Arecombinant reductase gene may or may not encode a protein comprising100% sequence identity with the amino acid sequence set forth in SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36. For example, arecombinant reductase gene may encode a protein having 100% sequenceidentity with a biologically-active portion of an amino acid sequenceset forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36.

A recombinant reductase gene may encode a reductase protein having,having at least, or having at most 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity with the amino acid sequence set forth in SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36, or abiologically-active portion thereof. A recombinant reductase gene mayencode a reductase protein having about, at least about, or at mostabout 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,100%, 100.1%, 100.2%, 100.3%, 100.4%, 100.5%, 100.6%, 100.7%, 100.8%,100.9%, 101%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%,150%, 160%, 170%, 180%, 190%, 200%, 220%, 240%, 260%, 280%, 300%, 320%,340%, 360%, 380%, or 400% reductase activity relative to a proteincomprising the amino acid sequence set forth in SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, or SEQ ID NO:36. A recombinant reductase gene mayencode a protein having, having at least, or having at most 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, or 500 contiguous amino acids starting at amino acid position1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500of the amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, or SEQ ID NO:36.

Substrates for the reductase protein may include any fatty acid from 14to 18 carbons long with a methylene substitution in the Δ9, Δ10, or Δ11position. The substrate may be 14, 15, 16, 17, or 18 carbons long, orany range derivable therein. The reductase protein may be capable ofcatalyzing the reduction of a methylene-substituted fatty acid substrateto a (methyl)lipid. The reductase protein, together with amethyltransferase protein, may be capable of catalyzing the productionof a methylated branch from any fatty acid from 14 to 18 carbons longwith an unsaturated double bond in the Δ9, Δ10, or Δ11 position,including fatty acids that are 14, 15, 16, 17, or 18 carbons long, orany range derivable therein.

In some embodiments, the recombinant reductase gene encodes a reductaseprotein that has specific amino acids unchanged from the amino acidsequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ IDNO:36. The unchanged amino acids can include 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids selected from 18, L22, F37, P38, R39, K41, G45, W46, P49, G144,C148, P149, E169, E171, L197, 1212, C249, H250, Y252, 1270, G275, L276,E283, A296, and A299 of Marinobacter hydrocarbonclasticus tmpA orcorresponding amino acids in tmpA from Desulfobacula balticum,Thiohalospira halophila, Desulfobacter curvatus, Desulfobacterphenolica, Desulfobacula toluolica, Desulfobacter postgatei, Halofilumochraceum, or Marinobacter aquaeolei, according to the alignment setforth in FIGS. 8A-D.

As used herein, the term “complementary” and derivatives thereof areused in reference to pairing of nucleic acids by the well-known rulesthat A pairs with T or U and C pairs with G. Complement can be “partial”or “complete”. In partial complement, only some of the nucleic acidbases are matched according to the base pairing rules; while in completeor total complement, all the bases are matched according to the pairingrule. The degree of complement between the nucleic acid strands may havesignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands as well known in the art. The efficiencyand strength of said hybridization depends upon the detection method.

Any nucleic acid that is referred to herein as having a certain percentsequence identity to a sequence set forth in a SEQ ID NO, includesnucleic acids that have the certain percent sequence identity to thecomplement of the sequence set forth in the SEQ ID NO.

d. Nucleic Acids Comprising a Recombinant Methyltransferase Gene and aRecombinant Reductase Gene

A nucleic acid may comprise both a recombinant methyltransferase geneand a recombinant reductase gene. The recombinant methyltransferase geneand the recombinant reductase gene may encode proteins from the samespecies or from different species.

A nucleic acid may comprise the nucleotide sequence of an expressionvector comprising a tmp operon that includes both a methyltransferasegene and a reductase gene. Such vectors may include pNC1071 (SEQ IDNO:39), which includes a Desulfobacter postgatei tmp operon; pNC1072(SEQ ID NO:40), which includes a Desulfobacula balticum tmp operon,pNC1073 (SEQ ID NO:41), which includes a Desulfobacula toluolica tmpoperon; pNC1074 (SEQ ID NO:42), which includes a Marinobacterhydrocarbonclasticus tmp operon; and pNC1076 (SEQ ID NO:43), whichincludes a Thiohalospira halophila tmp operon.

In some embodiments, the nucleic acid encodes a fusion protein thatincludes both a methyltransferase and a reductase or fragments thereof.In the context of the present invention, “fusion protein” means a singleprotein molecule containing two or more distinct proteins or fragmentsthereof, covalently linked via peptide bond in a single peptide chain.In some embodiments, the fusion protein comprises enzymatically activedomains from both a methyltransferase protein and a reductase protein.The nucleic acid may further encode a linker peptide between themethyltransferase and the reductase. In some embodiments, the linkerpeptide comprises the amino acid sequence AGGAEGGNGGGA (SEQ ID NO:44).The linker may comprise about or at least about 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, or 30 amino acids, or any range derivable therein. Thenucleic acid may comprise any of the methyltransferase and reductasegenes described herein, and the fusion protein encoded by the nucleicacid can comprise any of the methyltransferase and reductase proteinsdescribed herein, including biologically active fragments thereof. Insome embodiments, the fusion protein is a tmpA-B protein, in which thetmpA protein is closer to the N-terminus than the tmpB protein.

3. Compositions

Various aspects of the invention relate to compositions produced by thecells described herein. The composition may be an oil compositioncomprised of about, at least about, or at most about 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 100% lipids by weight. The composition may comprisebranched (methyl)lipids and/or exomethylene-substituted lipids. Thebranched (methyl)lipid may be a carboxylic acid (e.g., 10-methylstearicacid, 10-methylpalmitic acid, 12-methyloleic acid, 13-methyloleic acid,10-methyl-octadec-12-enoic acid), carboxylate (e.g., 10-methylstearate,10-methylpalmitate, 12-methyloleate, 13-methyloleate,10-methyl-octadec-12-enoate), ester (e.g., diacylglycerol,triacylglycerol, phospholipid), thioester (e.g., 10-methylstearyl CoA,10-methylpalmityl CoA, 12-methyloleoyl CoA, 13-methyloleoyl CoA,10-methyl-octadec-12-enoyl CoA), or amide. The exomethylene-substitutedlipid may be a carboxylic acid (e.g., 10-methylenestearic acid,10-methylenepalmitic acid, 12-methyleneoleic acid, 13-methyleneoleicacid, 10-methylene-octadec-12-enoic acid), carboxylate (e.g.,10-methylenestearate, 10-methylenepalmitate, 12-methyleneoleate,13-methyleneoleate, 10-methylene-octadec-12-enoate), ester (e.g.,diacylglycerol, triacylglycerol, phospholipid), thioester (e.g.,10-methylenestearyl CoA, 10-methylenepalmityl CoA, 12-methyleneoleoylCoA, 13-methyleneoleoyl CoA, 10-methylene-octadec-12-enoyl CoA), oramide. 10-methyl lipids, 10-methylene lipids, or both. It isspecifically contemplated that one or more of the above lipids may beexcluded from certain embodiments.

In some aspects, the composition is produced by cultivating a culturecomprising any of the cells described herein and recovering the oilcomposition from the cell culture. The cells in the culture may containany of the recombinant methyltransferase genes described herein and/orany of the recombinant reductase genes described herein. The culturemedium and conditions can be chosen based on the species of the cell tobe cultured and can be optimized to provide for maximal production ofthe desired lipid profile.

Various methods are known for recovering an oil composition from aculture of cells. For example, lipids, lipid derivatives, andhydrocarbons can be extracted with a hydrophobic solvent such as hexane.Lipids and lipid derivatives can also be extracted using liquefaction,oil liquefaction, and supercritical CO2 extraction. The recovery processmay include harvesting cultured cells, such as by filtration orcentrifugation, lysing cells to create a lysate, and extracting thelipid/hydrocarbon components using a hydrophobic solvent.

In addition to accumulating within cells, the lipids described hereinmay be secreted by the cells. In that case, a process for recovering thelipid may not require creating a lysate from the cells, but collectingthe secreted lipid from the culture medium. Thus, the compositionsdescribed herein may be made by culturing a cell that secretes one ofthe lipids described herein, such as a a linear fatty acid with a chainlength of 14-18 carbons with a methyl branch at the 49, MO, or 411position.

In some embodiments, the oil composition comprises about, at leastabout, or at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% by weight of a branched (methyl)lipid, such as a10-methyl fatty acid, or any range derivable therein. In someembodiments, 10-methyl fatty acids comprise about, at least about, or atmost about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% by weight of the fatty acids in the composition, or any rangederivable therein.

The amount of 10-methyl fatty acids in a cell can be optimized byvarious methods. For example, increasing the expression of tmpA and/ortmpB can increase the methyltransferase and/or reductase activity withinthe cell, which may lead to accumulation of greater amounts of branched(methyl lipids). One way this can be accomplished is by increasing thenumber of copies of the gene in the cell, such as by including the geneson high-copy-number plasmids. Additionally or alternatively, the tmpAand/or tmpB cells can be operably linked to a promoter that drives highlevels of expression.

4. Methods of Producing Branched (Methyl)Lipid

Various aspects of the invention relate to a method of producing abranched (methyl)lipid. The method may comprise incubating a cell orplurality of cells as described herein, supra, with media. The media mayoptionally be supplemented with an unbranched, unsaturated fatty acid,such as oleic acid, that serves as a substrate for methylation. Thesubstrate may include one or more fatty acids from 14 to 18 carbons longwith a double bond in the Δ9, Δ10, or Δ11 position. The substrate may be14, 15, 16, 17, or 18 carbons long, or any range derivable therein. Themedia may optionally be supplemented with methionine or s-adenosylmethionine, which may similarly serve as a substrate. Thus, the methodmay comprise contacting a cell or plurality of cells with oleic acid (orsome other substrate to be methylated), methionine, or both. The methodmay comprise incubating a cell or plurality of cells as describedherein, supra, in a bioreactor. The method may comprise recoveringlipids from the cells, such as by extraction with an organic solvent.

The method may comprise degumming the cell or plurality of cells, e.g.,to remove proteins. The method may comprise transesterification oresterification of the lipids of the cells. An alcohol such as methanolor ethanol may be used for transesterification or esterification, e.g.,thereby producing a fatty acid methyl ester or fatty acid ethyl ester.

EXAMPLES

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes only, and are not intended to limit the invention in anymanner. Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Identification of tmpB and tmpA Genes in Gammaproteobacteria

Select Gammaproteobacteria are known to produce branched 10-methyl fattyacids. The acetate-oxidizing, sulfate-reducing Desulfobacter bacteriawere reported to produce 10-methylhexadecanoic acid at 6%-24% of totalphospholipid-ester linked fatty acid content (Dowling, Microbiology132:1815-25 (1986)). Other reports of 10-methyl branched fatty acidproduction exist for bacteria in the Genus Marinobacter (Marquez, J.Syst. Evol. Microbiol. 55:1349-51 (2005); Huu, Int. J. Syst. Evol.Microbiol. 49:367-75 (1999); Gauthier, Int. J. Syst. Evol. Microbiol.42:568-76 (1992); Thiohalospira (Sorokin, Int. J. Syst. Evol.Microbiol., 58:2890-97 (2008)), Thiohalorhabdus (Sorokin, Int. J. Syst.Evol. Microbiol. 58:2890-97 (2008)), Desulfobacula, and Desulfotignum(Kuever, Int. J. Syst. Evol. Microbiol. 51:171-77 (2001)). However, nogenes or enzymes involved in Gammaproteobacteria 10-methyl fatty acidproduction have been described. In this Example, a pair ofphylogenetically and sequence-homology distinct genes present in certainGammaproteobacteria which direct production of 10-methyl fatty acids inheterologous hosts are described.

A list of Gammaproteobacteria that produce 10-methyl fatty acids andhave sequenced genomes was compiled from literature reports.Additionally, representative Gammaproteobacteria that are not reportedto produce 10-methyl fatty acids were included for comparison. Accordingto a biochemical study on the unrelated bacterium Mycobacterium phleiusing unpurified enzyme preparations, the first step of 10-methyl fattyacid synthesis occurs via a mechanism similar to cyclopropane fatty acidsynthesis and is followed by an enzymatic reduction step (Akamatsu, J.Biol. Chem. 245:701-08 (1970)). To find gene candidates responsible for10-methyl fatty acid production the Gammaproteobacteria genomes werescanned for homologs of E. coli cyclopropane fatty acyl phospholipidsynthase (cfa), which is responsible for methylation of unsaturatedfatty acids to produce cyclopropane fatty acids (Wang, Biochemistry31:11020-28 (1992); Taylor, Biochemistry 18:3292-3300 (1979)). This wasperformed using the NCBI BLAST protein analysis tool and the BioCycgenomic database (Caspi, Nucleic Acids Res. 40:D742-53 (2016)). Next,the cfa homologs were scanned for adjacent genes in an operon structurethat had homology to an oxidoreductase or electron transfer function.Interestingly, Gammaproteobacteria able to produce 10-methyl fatty acidsall possessed a gene operon (referred to herein as the tmp operon) witha cyclopropane fatty acid synthase gene homolog (referred to herein astmpB) and a gene with homology to a geranylgeranyl reductase (referredto herein as tmpA). These results are summarized in FIG. 2. It isunlikely tmpA is a true geranylgeranyl reductase since the enzyme isinvolved in chlorophyll and tocopherol biosynthesis, neither of whichchemicals the bacteria produce.

Example 2

E. coli Expression of the tmpB and tmpA Gene Products

To test if the tmp gene operon was responsible for Gammaproteobacteria10-methyl fatty acid production, the genes were designed in an E. coliexpression vector using the DNA manipulation software A Plasmid Editorand synthesized by Thermofisher Scientific-GeneArt. The native codonusage of the tmp genes was not changed. tmpB gene transcription wascontrolled using the constitutively active tac promoter (de Boer 1983),followed by the E. coli lacZ-lacY intergene linker region, the tmpAgene, and the trpT′ gene terminator (Wu 1981). These synthetic geneoperons were cloned into an E. coli expression vector containing theAmpR ampicillin resistance gene and the ColE1 origin of replication(FIG. 3A-3B). The plasmid vectors are named pNC1071 (SEQ ID NO:39),which includes the Desulfobacter postgatei tmp operon; pNC1072 (SEQ IDNO:40), which includes the Desulfobacula balticum tmp operon; pNC1073(SEQ ID NO:41), which includes the Desulfobacula toluolica tmp operon;pNC1074 (SEQ ID NO:42), which includes the Marinobacterhydrocarbonclasticus tmp operon; and pNC1076 (SEQ ID NO:43), whichincludes the Thiohalospira halophila tmp operon.

Plasmids pNC1071, pNC1072, pNC1073, pNC1074, pNC1076, and the controlplasmid pNC53 containing the AmpR gene, ColE1 origin, and tac promoterwere transformed into E. coli Top10 (Invitrogen) using a standardelectrotransformation protocol utilizing 50 μL suspended cells, 1 μL ofplasmid DNA at a concentration of 200 ng per μL, a 1 mm gapelectrotransformation cuvette, and a pulse with 1.8 kV voltage, 200Ω,and 25 μF with exponential decay and a time constant of approximately4.5 milliseconds. During the protocol cells were kept on ice and thecuvette was pre-chilled before pulsing with a Bio-Rad Gene PulserElectroporation System. After pulsing, cells were transferred to 1 mLSOC medium and incubated at 37° C. for 1 hour before plating on LB agarcontaining 100 μg per mL ampicillin antibiotic.

Single colonies from the transformation plates were chosen and grown in5 mL LB liquid media in 14 mL plastic falcon tubes overnight at 37° C.These were used to prepare freezer vials with 0.75 mL culture broth and0.75 mL of 50% glycerol/water which were stored at −80° C.

Fermentation studies were performed in 50 mL LB media with 100 μg per mLampicillin in 250 mL baffled shake flasks. 10 μL of frozen culture stockwas added to the media and the flask was incubated at 37° C. and shakenat 200 rpm in a New Brunswick orbital incubator for 24 hours. Cell wereharvested by centrifugation at 4000 rpm for 15 minutes in an Eppendorf5810 R clinical centrifuge, resuspended in 0.5 mL deionized water, andfrozen at −80° C.

FIG. 6 shows that E. coli transformed with pNC1071, pNC1073, pNC1074,and pNC1076, but not the empty vector control (pNC53) produced10-methylene hexadecenoic acid.

To test the acyl chain substrate range for the tmpB and tmpA enzymes, E.coli transformed with pNC1074 (M. hydrocarbonclausticus tmp operon) orpNC1076 (T. halophila tmp operon) were grown in LB media supplementedwith ampicillin and 100 mg/L of one of the fatty acids indicated inTable 1 below. After culturing, cells were harvested by centrifugation,washed with deionized water, resuspended in deionized water, and frozen.Cells were then lyophilized to dryness and used to perform aHCl-methanol catalyzed transesterification reaction to produce fattyacid methyl esters (FAME). These samples were dissolved in isooctane andinjected inot a gas chromatography system (Agilent Technologies)equipped with a flame ionization detector. Table 1 shows the percentageof each fatty acid that was converted to methylene- and methyl-branchedfatty acids.

TABLE 1 Fatty acid conversion to methylene and methyl branched fattyacids with E. coli expressing the tmpB and tmpA genes from M.hydrocarbonclasticus and T. halophila. E. coli + E. coli + pNC1074pNC1076 (M. hydrocarbonclausticus (T. halophila) tmpBA) tmpBA Fattypercent percent acid conversion conversion 12: 1Δ11  0%  0% 13: 1Δ12  0% 0% 14: 1Δ9  89% 95% 15: 1Δ10 86% 69% 16: 1Δ9  55% 95% 17: 1Δ10 36% 19%18: 1Δ6   0%  0% 18: 1Δ9  42% 47% 18: 1Δ11  9%  8% 19: 1Δ7   0%  0% 19:1Δ10  0%  0% 20: 1Δ5   0%  0% 20: 1Δ8   0%  0% 20: 1Δ11  0%  0% 22: 1Δ13 0%  0% 24: 1Δ15  0%  0%

As shown in Table 1, methylation occurred on fatty acids with 14, 15,16, 17, and 18 carbons, and on Δ9, Δ10, and Δ11 double bond positions.

Example 3 tmpB Gene Expression in Yeast

To test the production of 10-methylene fatty acids by the tmpB genes inthe yeast Saccharomyces cerevisiae and Yarrowia lipolytica, the genescontaining native bacterial codons were cloned into a standard Yarrowiaoverexpression vector. The vector contains a selectable NAT marker and a2μ origin of replication for high copy maintenance in Saccharomycescerevisiae. The resulting plasmids are pNC996 (Desulfobacter postgateitmpB), pNC998 (Desulfobacula balticum tmpB), pNC1000 (Desulfobaculatoluolica tmpB), pNC1002 (Marinobacter hydrocarbonclasticus tmpB),pNC1006 (Thiohalospira halophila tmpB). For Saccharomyces, plasmids weretransformed into NS20 by standard heat shock protocol. Single cells ofthe resulting transformations were selected and further grown in 96-wellshaking plates in YPD supplemented with 50 μg/mL Nourseothrycin for 2days at 30° C. For Yarrowia, plasmids were transformed into strainNS1009. Resulting transformed strains were grown in 96-well shakingplates in standard nitrogen limited media for 4 days at 30° C. For allyeast experiments, cell pellets were isolated by centrifugation andfreeze dried for fatty acid analysis by gas chromatography as performedfor E. coli samples. Total fatty acids were measured and the totalamount of C16 and C18 fatty acids containing the methylene intermediateswere quantified.

Results: Three tmpB genes produced 10-methylene fatty acids in NS20,Desulfobacula balticum, Marinobacter hydrocarbonclasticus, andThiohalospira halophila (FIG. 4). The tmpB genes from Marinobacterhydrocarbonclasticus, and Thiohalospira halophila were able to produce10-methylene fatty acids in Yarrowia lipolytica (FIG. 5).

Example 4 tmpB and tmpA Sequence Analysis

TmpB protein sequences encoded by the tmpB genes from Desulfobaculabalticum, Marinobacter hydrocarbonclasticus, Thiohalospira halophila,Desulfobacter curvatus, Desulfobacter phenolica, Desulfobaculatoluolica, Desulfobacter postgatei, Halofilum ochraceum, andMarinobacter aquaeolei were aligned with the cyclopropane fatty acidsynthase (Cfa) enzyme from Escherichia coli with the CLUSTAL OMEGAsoftware program (European Molecular Biology Laboratory, EMBL). FIGS.7A-D show the alignment of these protein sequences and indicates anumber of amino acids that are conserved in the tmsB protein sequencesbut not in the E. coli Cfa sequence. The following amino acids areconserved in the TmpB aligned proteins, but not present in the E. coliCfa protein: Y163, T175, R199, E211, G269, Y271, N313, N319, W389 (aminoacid number based on the M. hydrocarbonclasticus TmpB protein). Thepercent sequence identity of each of the aligned proteins as compared toM. hydrocarbonclasticus tmpB is indicated below:

% Identity of amino acid sequence Desulfobacula balticum TmpB 37%Thiohalospira halophila TmpB 58% Desulfobacter curvatus TmpB 43%Desulfobacter phenolica TmpB 39% Desulfobacula toluolica TmpB 39%Desulfobacter postgatei TmpB 43% Halofilum ochraceum TmpB 59%Marinobacter aquaeolei TmpB 88% Escherichia coli Cfa 46%

TmpA protein sequences encoded by the tmpA genes from Desulfobaculabalticum, Marinobacter hydrocarbonclasticus, Thiohalospira halophila,Desulfobacter curvatus, Desulfobacter phenolica, Desulfobaculatoluolica, Desulfobacter postgatei, Halofilum ochraceum, andMarinobacter aquaeolei were aligned with the Archaeoglobus fulgidusgeranylgeranyl reductase protein AF0464 with the CLUSTAL OMEGA softwareprogram (European Molecular Biology Laboratory, EMBL). FIGS. 8A-D showthe alignment of these protein sequences and indicates a number of aminoacids that are conserved in the tmsA protein sequences but not in theArchaeoglobus fulgidus geranylgeranyl reductase protein AF0464. Thefollowing amino acids are conserved in the TmpA aligned proteins, butnot present in the Archaeoglobus fulgidus geranylgeranyl reductaseprotein AF0464: 18, L22, F37, P38, R39, K41, G45, W46, P49, G144, C148,P149, E169, E171, L197, 1212, C249, H250, Y252, 1270, G275, L276, E283,A296, A299 (amino acid number based on the M. hydrocarbonclasticus TmpAprotein).

% Identity of amino acid sequence Desulfobacula balticum TmpA 33%Thiohalospira halophila TmpA 57% Desulfobacter curvatus TmpA 36%Desulfobacter phenolica TmpA 34% Desulfobacula toluolica TmpA 34%Desulfobacter postgatei TmpA 34% Halofilum ochraceum TmpA 64%Marinobacter aquaeolei TmpA 83% Archaeoglobus fulgidus AF0464 27%

1.-29. (canceled)
 30. A cell comprising an exogenous tmpB gene encodinga tmpB protein from a bacterium of the genus Desulfobacter,Desulfobacula, Marinobacter, Thiohalospira, Thiohalorhabdus,Desulfotignum, or Halofilum.
 31. The cell of claim 30, wherein the tmpBprotein is Desulfobacula balticum enzyme tmpB, Marinobacterhydrocarbonclasticus enzyme tmpB, Thiohalospira halophila enzyme tmpB,Desulfobacter curvatus enzyme tmpB, Desulfobacter phenolica enzyme tmpB,Desulfobacula toluolica enzyme tmpB, Desulfobacter postgatei enzymetmpB, Halojilum ochraceum enzyme tmpB, or Marinobacter aquaeolei enzymetmpB.
 32. The cell of claim 30, wherein the tmpB gene has at least 80%sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17.33. The cell of claim 30, wherein the tmpB gene is codon-optimized forexpression in yeast, algae, or plants.
 34. The cell of claim 30, whereinthe tmpB protein has at least 90% sequence identity to SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18.
 35. The cell of claim 30, furthercomprising a recombinant tmpA gene encoding a reductase tmpA proteinfrom a bacterium of the genus Desulfobacter, Desulfobacula,Marinobacter, Thiohalospira, Thiohalorhabdus, Desulfotignum, orHalofilum.
 36. The cell of claim 30, wherein the tmpA gene has at least80% sequence identity to SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQID NO:35.
 37. The cell of claim 35, wherein the tmpA protein is selectedfrom Desulfobacula balticum enzyme tmpA, Marinobacterhydrocarbonclasticus enzyme tmpA, Thiohalospira halophila enzyme tmpA,Desulfobacter curvatus enzyme tmpA, Desulfobacter phenolica enzyme tmpA,Desulfobacula toluolica enzyme tmpA, Desulfobacter postgatei enzymetmpA, Halofilum ochraceum enzyme tmpA, and Marinobacter aquaeolei enzymetmpA.
 38. The cell of claim 35, wherein the tmpA protein has at least90% sequence identity to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36.
 39. The cell of claim 35, wherein the tmpB gene and the tmpA geneare included in a single open reading frame encoding a fusion proteincomprising both the tmpB protein and the tmpA protein.
 40. The cell ofclaim 30, wherein the cell lacks an endogenous methyltransferase gene.41. The cell of claim 30, wherein the cell is a bacterial cell, a fungalcell, an algal cell, a mold cell, a plant cell, or a yeast cell.
 42. Thecell of claim 30, wherein the cell is a yeast cell.
 43. The cell ofclaim 30, wherein the cell is selected from the group consisting ofArxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillusterreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea,Cryptococcus albidus, Cryptococcus curvatus, Cryptococcusramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae,Cunninghamella echinulata, Cunninghamella japonica, Geotrichumfermentans, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromycesmarxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyceslipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierellaisabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii,Pichia guilliermondii, Pichia pastoris, Pichia stipites, Protothecazopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidiumtoruloides, Rhodosporidium paludigenum, Rhodotorula glutinis,Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomycespombe, Tremella enchepala, Trichosporon cutaneum, Trichosporonfermentans, Wickerhamomyces ciferrii, and Yarrowia lipolytica.
 44. Thecell of claim 30, wherein the cell is Yarrowia lipolytica.
 45. A methodof generating a transformed cell comprising introducing into a cell anexogenous tmpB gene encoding a tmpB protein from a bacterium of thegenus Desulfobacter, Desulfobacula, Marinobacter, Thiohalospira,Thiohalorhabdus, Desulfotignum, or Halofilum.
 46. The method of claim45, wherein the tmpB protein is Desulfobacula balticum enzyme tmpB,Marinobacter hydrocarbonclasticus enzyme tmpB, Thiohalospira halophilaenzyme tmpB, Desulfobacter curvatus enzyme tmpB, Desulfobacter phenolicaenzyme tmpB, Desulfobacula toluolica enzyme tmpB, Desulfobacterpostgatei enzyme tmpB, Halojilum ochraceum enzyme tmpB, or Marinobacteraquaeolei enzyme tmpB.
 47. The method of claim 45, wherein the tmpB genehas at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, or SEQ ID NO:17.
 48. The method of claim 45, wherein the tmpBgene is codon-optimized for expression in yeast, algae, or plants. 49.The method of claim 45, wherein the tmpB protein has at least 90%sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18.50. The method of claim 45, further comprising introducing into the cella recombinant tmpA gene encoding a reductase tmpA protein from abacterium of the genus Desulfobacter, Desulfobacula, Marinobacter,Thiohalospira, Thiohalorhabdus, Desulfotignum, or Halofilum.
 51. Themethod of claim 50, wherein the tmpA gene has at least 80% sequenceidentity to SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35. 52.The method of claim 50, wherein the tmpA protein is selected fromDesulfobacula balticum enzyme tmpA, Marinobacter hydrocarbonclasticusenzyme tmpA, Thiohalospira halophila enzyme tmpA, Desulfobacter curvatusenzyme tmpA, Desulfobacter phenolica enzyme tmpA, Desulfobaculatoluolica enzyme tmpA, Desulfobacter postgatei enzyme tmpA, Halofilumochraceum enzyme tmpA, and Marinobacter aquaeolei enzyme tmpA.
 53. Themethod of claim 50, wherein the tmpA protein has at least 90% sequenceidentity to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36. 54.The method of claim 50, wherein the tmpB gene and the tmpA gene areincluded in a single open reading frame encoding a fusion proteincomprising both the tmpB protein and the tmpA protein.
 55. The method ofclaim 45, wherein the cell lacks an endogenous methyltransferase gene.56. The method of claim 45, wherein the cell is a fungal cell, an algalcell, a mold cell, a plant cell, or a yeast cell.
 57. The method ofclaim 45, wherein the cell is a yeast cell.
 58. The method of claim 45,wherein the cell is selected from the group consisting of Arxulaadeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillusterreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea,Cryptococcus albidus, Cryptococcus curvatus, Cryptococcusramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae,Cunninghamella echinulata, Cunninghamella japonica, Geotrichumfermentans, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromycesmarxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyceslipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierellaisabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii,Pichia guilliermondii, Pichia pastoris, Pichia stipites, Protothecazopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidiumtoruloides, Rhodosporidium paludigenum, Rhodotorula glutinis,Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomycespombe, Tremella enchepala, Trichosporon cutaneum, Trichosporonfermentans, Wickerhamomyces ciferrii, and Yarrowia lipolytica.
 59. Themethod of claim 45, wherein the cell is Yarrowia lipolytica.
 60. Amethod of producing a branched (methyl)lipid or exomethylene-substitutedlipid, comprising contacting the cell of claim 30 with a substrate fattyacid, methionine, or both a substrate fatty acid and methionine, whereinthe substrate fatty acid comprises a fatty acid from 14 to 18 carbonslong with a double bond in the Δ9, Δ10, or Δ11 position.